Steroids 99 (2015) 108–112
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Review
Is there a role for estrogen activity assays? Recombinant cell bioassay for estrogen: Development and applications Karen Oerter Klein ⇑ University of California, San Diego, United States
a r t i c l e
i n f o
Article history: Received 12 May 2014 Accepted 10 June 2014 Available online 24 August 2014 Keywords: Puberty Turner syndrome Estradiol Bioassay Aromatase inhibition Menopause
a b s t r a c t There are many questions which cannot be answered without a very sensitive estradiol assay. A recombinant cell bioassay (RCBA) for estradiol was developed in 1994. The sensitivity of the bioassay is 0.02– 0.2 pg/ml (0.07–0.7 pmol/L), more than 20 times more sensitive than commercial RIAs and 10 times more sensitive than newer mass spectrometry assays. The RCBA for estradiol opened the door to study low levels of estradiol equivalents (EE) across the physiological spectrum of life from prepubertal children through menopause and across the spectrum from normal physiology, in boys as well as girls, to pathology, including: premature thelarche; estradiol suppression in children treated with GnRH analogues for precocious puberty; aromatase inhibition in boys with growth hormone deficiency; the differences between oral and transdermal routes of estrogen administration in girls with Turner’s syndrome; women with breast cancer treated with aromatase inhibitors; and women with urogenital atrophy treated with low dose vaginal estrogen. A bioassay also allows study of endocrine disruptors, like phytoestrogens and other environmental compounds, which are relevant to public health and alternative medicine options. This paper reviews the assay and the last 20 years of applications. A bioassay for estrogen has a role because measuring biological effect is theoretically useful, increasing the understanding of physiology in addition to biochemical levels, giving different information than other assays, and opening the door to measure very low levels of estrogen activity in both humans and the environment. Ó 2014 Elsevier Inc. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay and applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Estrogen bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. What are estrogen levels in prepubertal children? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. What are E2 levels in normal boys? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Are E2 levels in girls with premature thelarche higher than normal prepubertal girls? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. What are E2 levels in children with precocious puberty on treatment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Will long term estrogen suppression with anastrozole delay epiphyseal fusion in GH-treated adolescent males? . . . . . . . . . . . . . . . . . . 3.6. Do prepubertal girls with Turner’s have lower estrogen levels than age-matched girls without Turner syndrome?. . . . . . . . . . . . . . . . . 3.7. What is the optimal treatment to pubesce girls with Turner syndrome? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. How suppressed are estradiol levels in women with breast cancer on aromatase inhibitors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. What are estradiol levels in women treated with low-dose estradiol for urogenital atrophy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10. How quickly does estradiol recover after suppression in women treated with depot-leuprolide? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11. Which endocrine disruptors have estrogen-like effects? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
⇑ Address: Rady Children’s Hospital San Diego, 3020 Children’s Way, MC 5103, San Diego, CA 92123, United States. Tel.: +1 858 966 4032; fax: +1 858 966 6227. E-mail address: [email protected] http://dx.doi.org/10.1016/j.steroids.2014.08.004 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
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4.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Is there a role for estrogen activity Disclosure summary . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .
................................................................................. assays?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................................. .................................................................................
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1. Introduction There are many questions that could not previously be answered because of the lack of sensitivity of estradiol (E2) assays. This problem led to the development of a recombinant cell bioassay (RCBA) for estradiol in 1994 [1]. Over the past 20 years, this assay has been used in a wide range of clinical research settings from normal physiology in prepubertal children through menopause, and in pathological states and treatments involving very low levels of estrogen. The present paper reviews the assay development and the last 20 years of applications. 2. Assay and applications 2.1. Estrogen bioassay The bioassay for E2 uses a strain of Saccharomyces cerevisiae that is transformed with two plasmids. One plasmid contains the human estradiol receptor complementary DNA, and the other contains an estradiol response element upstream of the yeast iso-1-cytochrome C promoter fused to LACz, the structural gene for b-galactosidase. The transformed yeast is grown in selective media in the presence of extracted estradiol for 7 h. b-galactosidase activity is measured over time. The sensitivity of the bioassay ranges from 0.02 to 0.2 pg/ml (0.07–0.7 pmol/L). The intraassay and interassay coefficients of variation at 0.2 pg/ml (0.7 pmol/L) range from 10% to 50%. The standard curve is made by weighing estradiol and diluting into ethanol (Fig. 1). The assay is surprisingly specific for E2, with some cross-reactivity with estrogen metabolites. The assay correlates well with a standard RIA in the range measurable by RIA (Fig. 2). The limitations of the assay, and the major reasons why it is still a research assay rather than commercially available, include the expected variability of a bioassay and the extreme sensitivity of the yeast to the presence of estrogen. However, research use of the assay in the following situations shows the importance of continuing to improve methods for measuring both very low levels of E2 as well as estrogen bioactivity. All of the E2 levels reported in the following studies were measured using this RCBA. EE will be used as an abbreviation, since the bioassay more accurately represents estradiol equivalents.
Fig. 2. Linear regression of estrogen levels by RCBA vs. estradiol levels by RIA.
3. Applications 3.1. What are estrogen levels in prepubertal children? Prior to 1994, Bidlingmaier reported similar E2 levels in prepubertal boys and girls, but levels very close to the detection limit of the RIA used [2]. In 1994, we published much lower EE using the RCBA than previously thought, and levels significantly lower in prepubertal boys than girls [1] (Fig. 3). This is consistent with the differences in bone maturation and prepubertal growth observed between prepubertal girls and boys. 3.2. What are E2 levels in normal boys? Boys have much lower E2 levels than girls throughout life. We studied 23 normally growing boys and measured hormone levels and growth every 4 months for 5–8 years [3]. EE levels correlate strongly with testosterone levels and with peak growth velocity as boys progress through puberty (Fig. 4). This is consistent with the hypothesis that E2 augments skeletal growth in boys as well as girls. 3.3. Are E2 levels in girls with premature thelarche higher than normal prepubertal girls? Premature thelarche is the presence of breast tissue in girls prior to the onset of puberty, sometime less than age 8 years. We studied 20 girls with premature thelarche compared to 15 age-matched normal prepubertal girls (mean age 1.4 ± 0.5 year, range 0.5–3.0 year) [4]. EE levels were significantly higher in the girls with premature thelarche (data not shown). This supports the mechanism of premature thelarche being increased E2 levels rather than increased sensitivity of breast tissue to estrogens. 3.4. What are E2 levels in children with precocious puberty on treatment?
Fig. 1. Dose response curve of B-galactosidase activity vs. concentration of estradiol added to charcoal stripped plasma. Inset shows the low end of the curve.
Precocious puberty is defined as the onset of hypothalamicpituitary-gonadal axis activation prior to age 8 years in girls or
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n=20
25
(pmol/L)
Estradiol Equivalents
30
20 15
*
10
*
5 0
1
2
4
Dose (mcg/kg/d)
⁄
Fig. 3. EE in normal prepubertal boys and girls. Error bars represent ±1 SD. p < 0.05 compared to boys.
Fig. 5. EE in girls with precocious puberty assigned randomly to each of 3 GnRHa dose groups.
EE (pg/mL)
10 8 6 4 2 0
Baseline
3 Mo
6 Mo
9 Mo
12 Mo
Fig. 6. EE (solid bars) and testosterone levels (hatched bars) over 12 month study period in growth hormone deficient boys treated with anastrozole.
LH and EE were also measured in girls treated with depotleuprolide monthly injections [6]. LH and estradiol equivalents decreased on treatment (data not shown), but estradiol levels were still higher than in age matched normal prepubertal girls.
Fig. 4. Mean EE (solid bars), mean testosterone level (hatched bars), mean 24-h growth hormone level (striped bars), and growth velocity (solid line) in normal boys relative to year from peak growth velocity. EE are divided by 4 for purposes of presentation with the other hormones. Growth velocity is indicated on the same yaxis as hormone levels. Bars representing the same hormone but labeled with different letters are significantly different at the p < 0.05 level. Similar letters indicate no significant difference.
9 years in boys, with rapid progression and compromised adult height predictions. The treatment is to suppress puberty with GnRH analogues. Adequate treatment is assessed clinically, by measuring rate of bone maturation and by suppression of luteinizing hormone (LH) levels. E2 has not routinely been used to assess suppression due to the inability of E2 assays to reliably measure the low levels of E2 on treatment. EE were measured in 20 girls on treatment with the GnRH analogue, Deslorelin [5]. Estradiol equivalents were significantly higher in girls receiving a GnRHa dose of 1 mcg/kg/d vs 2 or 4 mcg/kg/d (Fig. 5). LH levels were not significantly higher in this cohort. This suggests the feasibility of measuring estradiol levels to monitor GnRHa treatment. The highest dose in this study, 4 mcg/kg/d, did not restore estradiol levels to the normal prepubertal range, which raises the question: Should the goal of GnRHa therapy be to suppress estradiol levels to the normal prepubertal range?
3.5. Will long term estrogen suppression with anastrozole delay epiphyseal fusion in GH-treated adolescent males? Mauras et al. studied GH-treated adolescent males given the aromatase inhibitor, anastrozole, to delay epiphyseal fusion and improve final adult height [7]. EE decreased while testosterone levels increased over 12 months of therapy (Fig 6). 3.6. Do prepubertal girls with Turner’s have lower estrogen levels than age-matched girls without Turner syndrome? Turner syndrome occurs in girls with a 45,X or 45,X mosaic karyotype. Girls have gonadal dysgenesis and phenotypic features which typically include: short stature, web neck, cubitus valgus, sexual infantilism (streak gonads), and delayed bone maturation.
Table 1 EE in girls with Turner syndrome receiving either oral or transdermal estrogen treatment.
Baseline 6 months 12 months *
Oral (pg/mL)
Transdermal
15 ± 5 183 ± 23 257 ± 19*
26 ± 9 141 ± 21 131 ± 25
P < 0.05 vs. transdermal group.
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The study design also led to the conclusion that the bioassay gives different information than the measurement of E2 by tandem mass spectrometry. 3.8. How suppressed are estradiol levels in women with breast cancer on aromatase inhibitors? Klein et al. reported suppression of EE in post-menopausal women treated with the aromatase inhibitor, letrozole. EE was 3fold lower than E2 measured by RIA [11]. (Fig 7) This study suggests that RIA at low levels measures non-specific blank values and that previously published estradiol results for post-menopausal women are erroneously high. It also suggests that letrozole may be potent at doses below 0.1 mg/d. 3.9. What are estradiol levels in women treated with low-dose estradiol for urogenital atrophy?
Fig. 7. Estradiol levels at baseline, 6 week of treatment, and 12 week of treatment, comparing the RCBA (solid bars) and the RIA (hatched bars), in women with breast cancer receiving the aromatase inhibitor, letrozole.
Seven women received 10 lm of estradiol vaginally twice per week and estradiol levels were measured over 24 h [12]. Indirect measures of an estrogen effect were seen as: (1) percent vaginal superficial cells were increased, (2) vaginal symptoms were decreased, and (3) vaginal pH decreased. Measurement of EE after administration of placebo cream revealed levels averaging 1–3 pg/ mL. After the initial insertion of vaginal cream, EE began to increase at 1 h and peaked at 4 h before returning to baseline at 8 h. (Fig 8) There was no significant decrease in LH levels providing further evidence of the minimal amounts of estrogen absorbed, and that very low dose vaginal estrogen improves symptoms without systemic affect, which could be detrimental in women with breast cancer. 3.10. How quickly does estradiol recover after suppression in women treated with depot-leuprolide? Women were given either 1 or 2 doses of Leuprolide acetate depot 3.75 mg [13]. EE was suppressed by 4 weeks after one dose and remained suppressed for 7 weeks, confirming the adequacy of monthly dosing and raising the question of whether less frequent dosing may be possible in some situations (data not shown). 3.11. Which endocrine disruptors have estrogen-like effects?
EE was lower in girls with Turner’s syndrome than age-matched girls without Turner syndrome (data not shown)[8]. This is consistent with the lack of normal ovarian function even before puberty in these girls. 3.7. What is the optimal treatment to pubesce girls with Turner syndrome? Taboada et al. studied estrogen replacement therapy in girls with Turner syndrome [9]. Ten girls were treated for 2 weeks with either low or high dose estrogen given either orally or transdermally (TD). EE was higher in girls receiving TD estrogen than in girls receiving oral estrogen in the doses studied (data not shown). EE was higher than E2 as determined by tandem mass spectrometry. The same group studied 40 girls over a 12-month period receiving either TD or oral estrogen replacement titrated to similar serum E2 levels as determined by tandem mass spectrometry [10]. EE results are shown in Table 1. E1 and E1S concentrations were much higher after oral dosing, suggesting that TD therapy results in a more physiological estrogen milieu than oral dosing.
Phytoestrogens are an area of interest in alternative medicine therapy and the study of endocrine disruptors in our environment has attracted wide spread interest. A bioassay can be used to
200 Estradiol Equivalents (pmol/L per mcgherb)
Fig. 8. Mean EE by week and time of day after low dose vaginal estrogen treatment. Values are mean + 1 SE. No differences are statistically significant.
Potency compared to E2
1/300
a,b
a,b
b
Soy
Clover Herb
Fo-Ti
160
1/80
c
120 80 40
a 0
Licorice
SoyA
Fig. 9. EE in five herbs. The bars represent mean + SD. The bars with different letters are significantly different from each other. P < 0.001 for SoyA; P < 0.05 for other herbs. The conversion factor to metric units is 1/3.671. Potency compared to estradiol is indicated at the top.
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measure estrogen activity in herbal products. We studied some common ingredients in alternative medicine options for hormone replacement therapy [14]. EE was measured in a variety of suspected phytoestrogens containing herbs and surprisingly high EE was found in Fo-Ti not previously reported (Fig 9). 4. Conclusion 4.1. Is there a role for estrogen activity assays? Measuring the biological effect of hormones is theoretically useful. Bioassays give the potential to understand physiology not just biochemical levels, and provides different information than other assays. The RCBA measures very low levels of estrogen activity in a wide range of physiologic and pathologic conditions across the lifespan in both genders, and allows for study of phytoestrogens and other compounds that may have estrogen like activity. Disclosure summary K.O.K. is a consultant for Endo Pharmaceuticals and AbbVie; has received grants from Pfizer and AbbVie; has been paid for participating in speaker’s bureaus and educational presentations for AbbVie; and has had travel/accommodations paid for by AbbVie and Endo Pharmaceuticals. References [1] Oerter KK, Baron J, Colli MJ, McDonnell DP, Cutler Jr GB. Estrogen levels in childhood determined by an ultra-sensitive recombinant cell bioassay. J Clin Invest 1994;94:2475–80. [2] Bidlingmaier F, Wagner-Barnack M, Butenandt O, Knorr D. Plasma estrogens in childhood and puberty under physiologic and pathologic conditions. Pediatr Res 1973;7(11):901–7.
[3] Klein KO, Martha Jr PM, Blizzard RM, Herbst T, Rogol AD. A longitudinal assessment of hormonal and physical alterations during normal puberty in boys II. Estrogen levels as determined by an ultrasensitive bioassay. J Clin Endocrinol Metab 1996;81:3203–7. [4] Klein KO, Mericq V, Brown JM, Larmore KA, Cabezas P, Cortinez A. Estrogen levels in girls with premature thelarche compared to normal prepubertal girls as determined by an ultrasensitive recombinant cell bioassay. J Pediatr 1999;134:190–2. [5] Klein KO, Baron J, Barnes KM, Pescovitz OH, Cutler Jr GB. Use of an ultrasensitive recombinant cell bioassay to determine estrogen levels in girls with precocious puberty treated with an LHRH agonist. J Clin Endocrinol Metab 1998;83:2387–9. [6] Kunz GJ, Sherman TI, Klein KO. LH and Estradiol suppression and growth in girls with central precocious puberty: Is more suppression better? J Pediatr Endocrinol Metab 2007;20:1189–98. [7] Mauras N, Welch S, Rini A, Klein KO. An open label 12-month pilot trial on the effects of the aromatase inhibitor anastrozole in growth hormone (GH)-treated GH deficient adolescent boys. J Pediatr Endocrinol Metab 2004;17:1597–606. [8] Wilson CA, Heinrichs C, Larmore KA, Craen M, Brown-Dawson J, Shaywitz S, Ross J, Klein KO. Estradiol levels in girls with Turner syndrome compared to normal prepubertal girls as determined by an ultrasensitive assay. J Pediatr Endocrinol Metab 2003;16:91–6. [9] Taboada M, Santen R, Lima J, Hossain J, Singh R, Klein KO, Mauras N. Pharmacokinetics and pharmacodynamics of oral and transdermal 17b estradiol in girls with Turner syndrome. J Clin Endocrinol Metab 2011;96(11):3502–10. [10] Torres-Santiago L, Mericq V, Taboada M, Unanue N, Klein KO, Singh R, Hossain J, Santen R, Ross JL, Mauras N. Metabolic effects of oral vs. transdermal 17-beta estradiol (E2): a randomized clinical trial in girls with Turner syndrome. J Clin Endocrinol Metab 2013;98:2716–24. [11] Oerter KK, Demers LM, Santner SJ, Baron J, Cutler GB, Santen RJ. Use of ultrasensitive recombinant cell bioassay to measure estrogen levels in women with breast cancer receiving the aromatase inhibitor, letrozole. J Clinical Endocrinol Metab 1995;80:2658–60. [12] Santen RJ, Pinkerton JV, Conaway MR, Ropka M, Wisniewski L, Demers L, Klein KO. Treatment of urogenital atrophy with low-dose estradiol: preliminary results. Menopause 2002;9(3):179–87. [13] Larmore KA, Klein KO. Estradiol suppression and recovery during leuprolide acetate treatment in women as determined weekly by an ultrasensitive recombinant cell bioassay. Gynecol. Endocrinol. 2000;14:405–10. [14] Klein KO, Janfaza M, Wong JA, Chang RJ. Estrogen bio-activity in Fo-Ti and other herbs used for their estrogen-like effects as determined by a recombinant cell bioassay. J Clin Endocrinol Metab 2003;88:4077–9.
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Review
Is there a role for estrogen activity assays? Recombinant cell bioassay for estrogen: Development and applications Karen Oerter Klein ⇑ University of California, San Diego, United States
a r t i c l e
i n f o
Article history: Received 12 May 2014 Accepted 10 June 2014 Available online 24 August 2014 Keywords: Puberty Turner syndrome Estradiol Bioassay Aromatase inhibition Menopause
a b s t r a c t There are many questions which cannot be answered without a very sensitive estradiol assay. A recombinant cell bioassay (RCBA) for estradiol was developed in 1994. The sensitivity of the bioassay is 0.02– 0.2 pg/ml (0.07–0.7 pmol/L), more than 20 times more sensitive than commercial RIAs and 10 times more sensitive than newer mass spectrometry assays. The RCBA for estradiol opened the door to study low levels of estradiol equivalents (EE) across the physiological spectrum of life from prepubertal children through menopause and across the spectrum from normal physiology, in boys as well as girls, to pathology, including: premature thelarche; estradiol suppression in children treated with GnRH analogues for precocious puberty; aromatase inhibition in boys with growth hormone deficiency; the differences between oral and transdermal routes of estrogen administration in girls with Turner’s syndrome; women with breast cancer treated with aromatase inhibitors; and women with urogenital atrophy treated with low dose vaginal estrogen. A bioassay also allows study of endocrine disruptors, like phytoestrogens and other environmental compounds, which are relevant to public health and alternative medicine options. This paper reviews the assay and the last 20 years of applications. A bioassay for estrogen has a role because measuring biological effect is theoretically useful, increasing the understanding of physiology in addition to biochemical levels, giving different information than other assays, and opening the door to measure very low levels of estrogen activity in both humans and the environment. Ó 2014 Elsevier Inc. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay and applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Estrogen bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. What are estrogen levels in prepubertal children? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. What are E2 levels in normal boys? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Are E2 levels in girls with premature thelarche higher than normal prepubertal girls? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. What are E2 levels in children with precocious puberty on treatment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Will long term estrogen suppression with anastrozole delay epiphyseal fusion in GH-treated adolescent males? . . . . . . . . . . . . . . . . . . 3.6. Do prepubertal girls with Turner’s have lower estrogen levels than age-matched girls without Turner syndrome?. . . . . . . . . . . . . . . . . 3.7. What is the optimal treatment to pubesce girls with Turner syndrome? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. How suppressed are estradiol levels in women with breast cancer on aromatase inhibitors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. What are estradiol levels in women treated with low-dose estradiol for urogenital atrophy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10. How quickly does estradiol recover after suppression in women treated with depot-leuprolide? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11. Which endocrine disruptors have estrogen-like effects? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
⇑ Address: Rady Children’s Hospital San Diego, 3020 Children’s Way, MC 5103, San Diego, CA 92123, United States. Tel.: +1 858 966 4032; fax: +1 858 966 6227. E-mail address: [email protected] http://dx.doi.org/10.1016/j.steroids.2014.08.004 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
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4.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Is there a role for estrogen activity Disclosure summary . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .
................................................................................. assays?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................................. .................................................................................
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1. Introduction There are many questions that could not previously be answered because of the lack of sensitivity of estradiol (E2) assays. This problem led to the development of a recombinant cell bioassay (RCBA) for estradiol in 1994 [1]. Over the past 20 years, this assay has been used in a wide range of clinical research settings from normal physiology in prepubertal children through menopause, and in pathological states and treatments involving very low levels of estrogen. The present paper reviews the assay development and the last 20 years of applications. 2. Assay and applications 2.1. Estrogen bioassay The bioassay for E2 uses a strain of Saccharomyces cerevisiae that is transformed with two plasmids. One plasmid contains the human estradiol receptor complementary DNA, and the other contains an estradiol response element upstream of the yeast iso-1-cytochrome C promoter fused to LACz, the structural gene for b-galactosidase. The transformed yeast is grown in selective media in the presence of extracted estradiol for 7 h. b-galactosidase activity is measured over time. The sensitivity of the bioassay ranges from 0.02 to 0.2 pg/ml (0.07–0.7 pmol/L). The intraassay and interassay coefficients of variation at 0.2 pg/ml (0.7 pmol/L) range from 10% to 50%. The standard curve is made by weighing estradiol and diluting into ethanol (Fig. 1). The assay is surprisingly specific for E2, with some cross-reactivity with estrogen metabolites. The assay correlates well with a standard RIA in the range measurable by RIA (Fig. 2). The limitations of the assay, and the major reasons why it is still a research assay rather than commercially available, include the expected variability of a bioassay and the extreme sensitivity of the yeast to the presence of estrogen. However, research use of the assay in the following situations shows the importance of continuing to improve methods for measuring both very low levels of E2 as well as estrogen bioactivity. All of the E2 levels reported in the following studies were measured using this RCBA. EE will be used as an abbreviation, since the bioassay more accurately represents estradiol equivalents.
Fig. 2. Linear regression of estrogen levels by RCBA vs. estradiol levels by RIA.
3. Applications 3.1. What are estrogen levels in prepubertal children? Prior to 1994, Bidlingmaier reported similar E2 levels in prepubertal boys and girls, but levels very close to the detection limit of the RIA used [2]. In 1994, we published much lower EE using the RCBA than previously thought, and levels significantly lower in prepubertal boys than girls [1] (Fig. 3). This is consistent with the differences in bone maturation and prepubertal growth observed between prepubertal girls and boys. 3.2. What are E2 levels in normal boys? Boys have much lower E2 levels than girls throughout life. We studied 23 normally growing boys and measured hormone levels and growth every 4 months for 5–8 years [3]. EE levels correlate strongly with testosterone levels and with peak growth velocity as boys progress through puberty (Fig. 4). This is consistent with the hypothesis that E2 augments skeletal growth in boys as well as girls. 3.3. Are E2 levels in girls with premature thelarche higher than normal prepubertal girls? Premature thelarche is the presence of breast tissue in girls prior to the onset of puberty, sometime less than age 8 years. We studied 20 girls with premature thelarche compared to 15 age-matched normal prepubertal girls (mean age 1.4 ± 0.5 year, range 0.5–3.0 year) [4]. EE levels were significantly higher in the girls with premature thelarche (data not shown). This supports the mechanism of premature thelarche being increased E2 levels rather than increased sensitivity of breast tissue to estrogens. 3.4. What are E2 levels in children with precocious puberty on treatment?
Fig. 1. Dose response curve of B-galactosidase activity vs. concentration of estradiol added to charcoal stripped plasma. Inset shows the low end of the curve.
Precocious puberty is defined as the onset of hypothalamicpituitary-gonadal axis activation prior to age 8 years in girls or
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n=20
25
(pmol/L)
Estradiol Equivalents
30
20 15
*
10
*
5 0
1
2
4
Dose (mcg/kg/d)
⁄
Fig. 3. EE in normal prepubertal boys and girls. Error bars represent ±1 SD. p < 0.05 compared to boys.
Fig. 5. EE in girls with precocious puberty assigned randomly to each of 3 GnRHa dose groups.
EE (pg/mL)
10 8 6 4 2 0
Baseline
3 Mo
6 Mo
9 Mo
12 Mo
Fig. 6. EE (solid bars) and testosterone levels (hatched bars) over 12 month study period in growth hormone deficient boys treated with anastrozole.
LH and EE were also measured in girls treated with depotleuprolide monthly injections [6]. LH and estradiol equivalents decreased on treatment (data not shown), but estradiol levels were still higher than in age matched normal prepubertal girls.
Fig. 4. Mean EE (solid bars), mean testosterone level (hatched bars), mean 24-h growth hormone level (striped bars), and growth velocity (solid line) in normal boys relative to year from peak growth velocity. EE are divided by 4 for purposes of presentation with the other hormones. Growth velocity is indicated on the same yaxis as hormone levels. Bars representing the same hormone but labeled with different letters are significantly different at the p < 0.05 level. Similar letters indicate no significant difference.
9 years in boys, with rapid progression and compromised adult height predictions. The treatment is to suppress puberty with GnRH analogues. Adequate treatment is assessed clinically, by measuring rate of bone maturation and by suppression of luteinizing hormone (LH) levels. E2 has not routinely been used to assess suppression due to the inability of E2 assays to reliably measure the low levels of E2 on treatment. EE were measured in 20 girls on treatment with the GnRH analogue, Deslorelin [5]. Estradiol equivalents were significantly higher in girls receiving a GnRHa dose of 1 mcg/kg/d vs 2 or 4 mcg/kg/d (Fig. 5). LH levels were not significantly higher in this cohort. This suggests the feasibility of measuring estradiol levels to monitor GnRHa treatment. The highest dose in this study, 4 mcg/kg/d, did not restore estradiol levels to the normal prepubertal range, which raises the question: Should the goal of GnRHa therapy be to suppress estradiol levels to the normal prepubertal range?
3.5. Will long term estrogen suppression with anastrozole delay epiphyseal fusion in GH-treated adolescent males? Mauras et al. studied GH-treated adolescent males given the aromatase inhibitor, anastrozole, to delay epiphyseal fusion and improve final adult height [7]. EE decreased while testosterone levels increased over 12 months of therapy (Fig 6). 3.6. Do prepubertal girls with Turner’s have lower estrogen levels than age-matched girls without Turner syndrome? Turner syndrome occurs in girls with a 45,X or 45,X mosaic karyotype. Girls have gonadal dysgenesis and phenotypic features which typically include: short stature, web neck, cubitus valgus, sexual infantilism (streak gonads), and delayed bone maturation.
Table 1 EE in girls with Turner syndrome receiving either oral or transdermal estrogen treatment.
Baseline 6 months 12 months *
Oral (pg/mL)
Transdermal
15 ± 5 183 ± 23 257 ± 19*
26 ± 9 141 ± 21 131 ± 25
P < 0.05 vs. transdermal group.
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The study design also led to the conclusion that the bioassay gives different information than the measurement of E2 by tandem mass spectrometry. 3.8. How suppressed are estradiol levels in women with breast cancer on aromatase inhibitors? Klein et al. reported suppression of EE in post-menopausal women treated with the aromatase inhibitor, letrozole. EE was 3fold lower than E2 measured by RIA [11]. (Fig 7) This study suggests that RIA at low levels measures non-specific blank values and that previously published estradiol results for post-menopausal women are erroneously high. It also suggests that letrozole may be potent at doses below 0.1 mg/d. 3.9. What are estradiol levels in women treated with low-dose estradiol for urogenital atrophy?
Fig. 7. Estradiol levels at baseline, 6 week of treatment, and 12 week of treatment, comparing the RCBA (solid bars) and the RIA (hatched bars), in women with breast cancer receiving the aromatase inhibitor, letrozole.
Seven women received 10 lm of estradiol vaginally twice per week and estradiol levels were measured over 24 h [12]. Indirect measures of an estrogen effect were seen as: (1) percent vaginal superficial cells were increased, (2) vaginal symptoms were decreased, and (3) vaginal pH decreased. Measurement of EE after administration of placebo cream revealed levels averaging 1–3 pg/ mL. After the initial insertion of vaginal cream, EE began to increase at 1 h and peaked at 4 h before returning to baseline at 8 h. (Fig 8) There was no significant decrease in LH levels providing further evidence of the minimal amounts of estrogen absorbed, and that very low dose vaginal estrogen improves symptoms without systemic affect, which could be detrimental in women with breast cancer. 3.10. How quickly does estradiol recover after suppression in women treated with depot-leuprolide? Women were given either 1 or 2 doses of Leuprolide acetate depot 3.75 mg [13]. EE was suppressed by 4 weeks after one dose and remained suppressed for 7 weeks, confirming the adequacy of monthly dosing and raising the question of whether less frequent dosing may be possible in some situations (data not shown). 3.11. Which endocrine disruptors have estrogen-like effects?
EE was lower in girls with Turner’s syndrome than age-matched girls without Turner syndrome (data not shown)[8]. This is consistent with the lack of normal ovarian function even before puberty in these girls. 3.7. What is the optimal treatment to pubesce girls with Turner syndrome? Taboada et al. studied estrogen replacement therapy in girls with Turner syndrome [9]. Ten girls were treated for 2 weeks with either low or high dose estrogen given either orally or transdermally (TD). EE was higher in girls receiving TD estrogen than in girls receiving oral estrogen in the doses studied (data not shown). EE was higher than E2 as determined by tandem mass spectrometry. The same group studied 40 girls over a 12-month period receiving either TD or oral estrogen replacement titrated to similar serum E2 levels as determined by tandem mass spectrometry [10]. EE results are shown in Table 1. E1 and E1S concentrations were much higher after oral dosing, suggesting that TD therapy results in a more physiological estrogen milieu than oral dosing.
Phytoestrogens are an area of interest in alternative medicine therapy and the study of endocrine disruptors in our environment has attracted wide spread interest. A bioassay can be used to
200 Estradiol Equivalents (pmol/L per mcgherb)
Fig. 8. Mean EE by week and time of day after low dose vaginal estrogen treatment. Values are mean + 1 SE. No differences are statistically significant.
Potency compared to E2
1/300
a,b
a,b
b
Soy
Clover Herb
Fo-Ti
160
1/80
c
120 80 40
a 0
Licorice
SoyA
Fig. 9. EE in five herbs. The bars represent mean + SD. The bars with different letters are significantly different from each other. P < 0.001 for SoyA; P < 0.05 for other herbs. The conversion factor to metric units is 1/3.671. Potency compared to estradiol is indicated at the top.
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measure estrogen activity in herbal products. We studied some common ingredients in alternative medicine options for hormone replacement therapy [14]. EE was measured in a variety of suspected phytoestrogens containing herbs and surprisingly high EE was found in Fo-Ti not previously reported (Fig 9). 4. Conclusion 4.1. Is there a role for estrogen activity assays? Measuring the biological effect of hormones is theoretically useful. Bioassays give the potential to understand physiology not just biochemical levels, and provides different information than other assays. The RCBA measures very low levels of estrogen activity in a wide range of physiologic and pathologic conditions across the lifespan in both genders, and allows for study of phytoestrogens and other compounds that may have estrogen like activity. Disclosure summary K.O.K. is a consultant for Endo Pharmaceuticals and AbbVie; has received grants from Pfizer and AbbVie; has been paid for participating in speaker’s bureaus and educational presentations for AbbVie; and has had travel/accommodations paid for by AbbVie and Endo Pharmaceuticals. References [1] Oerter KK, Baron J, Colli MJ, McDonnell DP, Cutler Jr GB. Estrogen levels in childhood determined by an ultra-sensitive recombinant cell bioassay. J Clin Invest 1994;94:2475–80. [2] Bidlingmaier F, Wagner-Barnack M, Butenandt O, Knorr D. Plasma estrogens in childhood and puberty under physiologic and pathologic conditions. Pediatr Res 1973;7(11):901–7.
[3] Klein KO, Martha Jr PM, Blizzard RM, Herbst T, Rogol AD. A longitudinal assessment of hormonal and physical alterations during normal puberty in boys II. Estrogen levels as determined by an ultrasensitive bioassay. J Clin Endocrinol Metab 1996;81:3203–7. [4] Klein KO, Mericq V, Brown JM, Larmore KA, Cabezas P, Cortinez A. Estrogen levels in girls with premature thelarche compared to normal prepubertal girls as determined by an ultrasensitive recombinant cell bioassay. J Pediatr 1999;134:190–2. [5] Klein KO, Baron J, Barnes KM, Pescovitz OH, Cutler Jr GB. Use of an ultrasensitive recombinant cell bioassay to determine estrogen levels in girls with precocious puberty treated with an LHRH agonist. J Clin Endocrinol Metab 1998;83:2387–9. [6] Kunz GJ, Sherman TI, Klein KO. LH and Estradiol suppression and growth in girls with central precocious puberty: Is more suppression better? J Pediatr Endocrinol Metab 2007;20:1189–98. [7] Mauras N, Welch S, Rini A, Klein KO. An open label 12-month pilot trial on the effects of the aromatase inhibitor anastrozole in growth hormone (GH)-treated GH deficient adolescent boys. J Pediatr Endocrinol Metab 2004;17:1597–606. [8] Wilson CA, Heinrichs C, Larmore KA, Craen M, Brown-Dawson J, Shaywitz S, Ross J, Klein KO. Estradiol levels in girls with Turner syndrome compared to normal prepubertal girls as determined by an ultrasensitive assay. J Pediatr Endocrinol Metab 2003;16:91–6. [9] Taboada M, Santen R, Lima J, Hossain J, Singh R, Klein KO, Mauras N. Pharmacokinetics and pharmacodynamics of oral and transdermal 17b estradiol in girls with Turner syndrome. J Clin Endocrinol Metab 2011;96(11):3502–10. [10] Torres-Santiago L, Mericq V, Taboada M, Unanue N, Klein KO, Singh R, Hossain J, Santen R, Ross JL, Mauras N. Metabolic effects of oral vs. transdermal 17-beta estradiol (E2): a randomized clinical trial in girls with Turner syndrome. J Clin Endocrinol Metab 2013;98:2716–24. [11] Oerter KK, Demers LM, Santner SJ, Baron J, Cutler GB, Santen RJ. Use of ultrasensitive recombinant cell bioassay to measure estrogen levels in women with breast cancer receiving the aromatase inhibitor, letrozole. J Clinical Endocrinol Metab 1995;80:2658–60. [12] Santen RJ, Pinkerton JV, Conaway MR, Ropka M, Wisniewski L, Demers L, Klein KO. Treatment of urogenital atrophy with low-dose estradiol: preliminary results. Menopause 2002;9(3):179–87. [13] Larmore KA, Klein KO. Estradiol suppression and recovery during leuprolide acetate treatment in women as determined weekly by an ultrasensitive recombinant cell bioassay. Gynecol. Endocrinol. 2000;14:405–10. [14] Klein KO, Janfaza M, Wong JA, Chang RJ. Estrogen bio-activity in Fo-Ti and other herbs used for their estrogen-like effects as determined by a recombinant cell bioassay. J Clin Endocrinol Metab 2003;88:4077–9.
